Case studies of ends‐out gene targeting in Drosophila

Ends‐in and ends‐out gene replacement approaches have been successfully used to disrupt Drosophila genes involved in a variety of biological processes. These methods combine double‐strand breaks and homologous recombination to replace a targeted chromosome region with a designed DNA sequence. Unfortunately, these methods require large numbers of single animal crosses, making them both time consuming and labor intensive. Here, we designed a single complete targeting vector for use in a mass crossing ends‐out gene targeting study. Importantly, our gene targeting method included a balancer chromosome to block endogenous homologous chromosome pairing and to promote pairing between the foreign targeting DNA fragment and the targeted chromosome. This technique provided successful and efficient gene replacement, greatly facilitating the gene knockout procedure. genesis 47:305–308, 2009. © 2009 Wiley‐Liss, Inc.     

Reverse genetics system for human rotaviruses

A reverse genetics technology is an incredibly useful technique both for a proper understanding of different aspects of virus biology and for the generation of complementary DNA (cDNA)-derived infectious viruses, which can act as safe and effective vaccines and viral vectors. Rotaviruses (RVAs), especially human RVAs (HuRVAs), had been very refractory to this technology until very recently. Here, we describe the historical background of the development of a long-awaited HuRVA reverse genetics system, culminating in the generation of replicative HuRVAs entirely from cloned cDNAs.     

Average gene length is highly conserved in prokaryotes and eukaryotes and diverges only between the two kingdoms

The average length of genes in a eukaryote is larger than in a prokaryote, implying that evolution of complexity is related to change of gene lengths. Here, we show that although the average lengths of genes in prokaryotes and eukaryotes are much different, the average lengths of genes are highly conserved within either of the two kingdoms. This suggests that natural selection has clearly set a strong limitation on gene elongation within the kingdom. Furthermore, the average gene size adds another distinct characteristic for the discrimination between the two kingdoms of organisms.     

Planctomycetes and eukaryotes: a case of analogy not homology

Planctomycetes, Verrucomicrobia and Chlamydia are prokaryotic phyla, sometimes grouped together as the PVC superphylum of eubacteria. Some PVC species possess interesting attributes, in particular, internal membranes that superficially resemble eukaryotic endomembranes. Some biologists now claim that PVC bacteria are nucleus-bearing prokaryotes and are considered evolutionary intermediates in the transition from prokaryote to eukaryote. PVC prokaryotes do not possess a nucleus and are not intermediates in the prokaryote-to-eukaryote transition. Here we summarise the evidence that shows why all of the PVC traits that are currently cited as evidence for aspiring eukaryoticity are either analogous (the result of convergent evolution), not homologous, to eukaryotic traits; or else they are the result of horizontal gene transfers.     

Reverse Genetic Approaches for Functional Genomics of Rice

T-DNA and transposable elements e.g., Ds and Tos17, are used to generate a large number of insertional mutant lines in rice. Some carry the GUS or GFP reporter for gene trap or enhancer trap. These reporter systems are valuable for identifying tissue- or organ-preferential genes. Activation tagging lines have also been generated for screening mutants and isolating mutagenized genes. To utilize these resources more efficiently, tagged lines have been produced for reverse genetic approaches. DNA pools of the T-DNA tagged lines and Tos17 lines have been prepared for PCR screening of insertional mutants in a given gene. Tag end sequences (TES) of the inserts have also been produced. TES databases are beneficial for analyzing the function of a large number of rice genes.     

基因打靶技术:开启遗传学新纪元

基因打靶技术作为最有效的定向修饰小鼠基因的技术手段在揭示基因的生理功能、研究人类疾病的遗传机制以及寻找新的药物靶标的过程中发挥着重要的作用。近年来, 随着条件基因打靶技术的发展使基因失活可以限制在特定时段特定组织或细胞内。文章将主要介绍基因打靶技术的发展简史、近期进展以及在其他模式动物中的应用。     

微生物基因组简化的研究进展

微生物基因组简化是合成生物学研究热点之一。基因组的适度精简可使细胞代谢途径得以优化,改善细胞对底物、能量的利用效率,大大提高细胞生理性能的预测性和可控性。基因组简化细胞将为生物技术的应用提供理想的底盘细胞。同顾了构建基因组简化细胞的研究策略、研究方法及一些模式生物相关研究进展,总结了基因组简化研究所面临的问题及解决办法,对基因组减小化研究发展趋势前景进行了展望。     

Smad3基因剔除小鼠的繁殖与基因型鉴定

目的为进一步深入研究Smad3基因在脊椎动物发育中的重要作用,对Smad3基因剔除小鼠进行保种和繁育研究.方法采用基因剔除杂合子小鼠进行保种,通过PCR和Southern杂交对杂合子小鼠交配所产生的后代进行基因型鉴定,纯合子小鼠和野生型小鼠用于表型分析,杂合子小鼠用于留种和繁殖生产.结果采用PCR方法对278只子代小鼠进行了基因型鉴定,83只为野生型,133只为杂合子,62只为纯合子.结论Smad3基因剔除突变能稳定遗传.采用杂合子小鼠保种,子代小鼠三种基因型比例符合孟德尔遗传定律.     

Generation of Ppp2Ca and Ppp2Cb conditional null alleles in mouse

Protein phosphatase 2A (PP2A) is one of the most abundant serine/threonine phosphatases, with a critical role in embryonic development and human disease. There are two isoforms of the catalytic subunit of PP2A, Ppp2ca and Ppp2cb. Null mutation of Ppp2ca leads to early embryonic lethality at E6.5, hindering functional study of PP2A beyond this stage. We generated conditional null alleles of Ppp2ca and Ppp2cb by flanking with loxP sites exons 3 to 5 of Ppp2ca and exon 3 of Ppp2cb. Ppp2ca(fl/fl) mice did not display any visible phenotype. Homozygous mutants in which Cre-mediated excision resulted in global deletion of Ppp2ca displayed embryonic lethality and developmental defects similar to those previously reported. Ppp2cb(Δ/Δ) mice generated by the same strategy did not display any obvious morphological or physiological defects. These mouse strains can serve as important genetic tools to study the roles of PP2A during development and disease in a spatial- or temporal-specific manner.     

Generation of Fgfr3 Conditional Knockout Mice

Fibroblast growth factor receptor 3 (FGFR3), highly conserved in both humans and murine, is one of key tyrosine kinase receptors for FGF. FGFR3 is expressed in different tissues, including cartilage, brain, kidney, and intestine at different development stages. Conventional knockout of Fgfr3 alleles leads to short life span, and overgrowth of bone. In clinic, human FGFR3 mutations are responsible for three different types of chondrodysplasia syndromes including achondroplasia (ACH), hypochondroplasia (HCH) and thanatophoric dysplasia (TD). For better understanding of the roles of FGFR3 in different tissues at different stages of development and in pathological conditions, we generated Fgfr3 conditional knockout mice in which loxp sites flank exons 9-10 in the Fgfr3 allele. We also demonstrated that Cre-mediated recombination using Col2a1-Cre, a Cre line expressed in chondrocyte during bone development, results in specific deletion of the gene in tissues containing cartilage. This animal model will be useful to study distinct roles of FGFR3 in different tissues at different ages.     

Cre/lox位点特异性重组系统在高等真核生物中的研究进展

来自于P1噬菌体的Cre/lox系统通过位点特异性重组可以迅速而有效地实现各种生理环境下的基因定点插入、删除、替换和倒位等操作。Cre/lox系统作为目前基因打靶技术的核心工具,已被广泛应用于拟南芥、水稻、小鼠、果蝇、斑马鱼等高等真核模式生物。文章较为全面地介绍了Cre/lox系统的基本概况及其在高等真核生物中的应用,讨论了Cre/lox系统在研究中存在的主要问题和今后的发展方向,为利用该系统在不同高等生物中进行基因操作提供有用的参考。     

Cre/lox位点特异性重组系统在高等真核生物中的研究进展

来自于P1噬菌体的Cre/lox系统通过位点特异性重组可以迅速而有效地实现各种生理环境下的基因定点插入、删除、替换和倒位等操作。Cre/lox系统作为目前基因打靶技术的核心工具, 已被广泛应用于拟南芥、水稻、小鼠、果蝇、斑马鱼等高等真核模式生物。文章较为全面地介绍了Cre/lox系统的基本概况及其在高等真核生物中的应用, 讨论了Cre/lox系统在研究中存在的主要问题和今后的发展方向, 为利用该系统在不同高等生物中进行基因操作提供有用的参考。     

Mouse genetics in the 21st century: using gene targeting to create a cornucopia of mouse mutants possessing precise genetic modifications

Over 1500 mouse mutants have been identified, but few of the genes responsible for the defects have been identified. Recent developments in the area of gene targeting are revolutionizing the field of mouse genetics and our understanding of numerous genes, including those thought to be involved in cell proliferation and differentiation. Gene targeting was developed as a method for producing a predetermined mutation in a specific endogenous gene. Advances in the design of targeting vectors and in the use of embryonic stem cells have permitted the production of numerous mutant mice with null mutations in specific genes. These mutant mice will be critical for investigating thein vivo functions of many genes that have been cloned in recent years. This review discusses a wide range of new developments in the field of gene targeting with a focus on issues to be considered by those planning to use this new technology. It also examines some of the lessons learned from recent gene targeting studies and discusses different applications of the technology that are likely to generate scores of new animal models for a wide range of human diseases.Abbreviations ES embryonic stem - neo^r neomycin resistance gene - HSV herpes simplex virus - tk thymidine kinase gene - PCR polymerase chain reaction - LIF leukemia inhibitory factor - LTP long-term potentiation - Rb retinoblastoma gene product - CF cystic fibrosis     

Suppression of OsRAD51D results in defects in reproductive development in rice (Oryza sativa L.)

The cellular roles of RAD51 paralogs in somatic and reproductive growth have been extensively described in a wide range of animal systems and, to a lesser extent, in Arabidopsis, a dicot model plant. Here, the OsRAD51D gene was identified and characterized in rice (Oryza sativa L.), a monocot model crop. In the rice genome, three alternative OsRAD51D mRNA splicing variants, OsRAD51D.1, OsRAD51D.2, and OsRAD51D.3, were predicted. Yeast two‐hybrid studies, however, showed that only OsRAD51D.1 interacted with OsRAD51B and OsRAD51C paralogs, suggesting that OsRAD51D.1 is a functional OsRAD51D protein in rice. Loss‐of‐function osrad51d mutant rice plants displayed normal vegetative growth. However, the mutant plants were defective in reproductive growth, resulting in sterile flowers. Homozygous osrad51d mutant flowers exhibited impaired development of lemma and palea and contained unusual numbers of stamens and stigmas. During early meiosis, osrad51d pollen mother cells (PMCs) failed to form normal homologous chromosome pairings. In subsequent meiotic progression, mutant PMCs represented fragmented chromosomes. The osrad51d pollen cells contained numerous abnormal micro‐nuclei that resulted in malfunctioning pollen. The abnormalities of heterozygous mutant and T2 Ubi:RNAi‐OsRAD51D RNAi‐knock‐down transgenic plants were intermediate between those of wild type and homozygous mutant plants. The osrad51d and Ubi:RNAi‐OsRAD51D plants contained longer telomeres compared with wild type plants, indicating that OsRAD51D is a negative factor for telomere lengthening. Overall, these results suggest that OsRAD51D plays a critical role in reproductive growth in rice. This essential function of OsRAD51D is distinct from Arabidopsis, in which AtRAD51D is not an essential factor for meiosis or reproductive development.     

Targeted disruption of exogenous EGFP gene in medaka using zinc-finger nucleases

Zinc-finger nucleases (ZFNs) are artificial enzymes that create site-specific double-strand breaks and thereby induce targeted genome editing. Here, we demonstrated successful gene disruption in somatic and germ cells of medaka (Oryzias latipes) using ZFN to target exogenous EGFP genes. Embryos that were injected with an RNA sequence pair coding for ZFNs showed mosaic loss of green fluorescent protein fluorescence in skeletal muscle. A number of mutations that included both deletions and insertions were identified within the ZFN target site in each embryo, whereas no mutations were found at the non-targeted sites. In addition, ZFN-induced mutations were introduced in germ cells and efficiently transmitted to the next generation. The mutation frequency varied (6-100%) in the germ cells from each founder, and a founder carried more than two types of mutation in germ cells. Our results have introduced the possibility of targeted gene disruption and reverse genetics in medaka.     

Genetic engineering of rotaviruses by reverse genetics

The rotavirus genome is composed of 11 gene segments of dsRNA. A recent breakthrough in the field of rotaviruses is the development of a reverse genetics system for generating recombinant rotaviruses possessing a gene segment derived from cloned cDNA. Although this approach is a helper virus‐driven system that is technically limited and gives low levels of recombinant viruses, it allows alteration of the rotavirus genome, thus contributing to our understanding of these medically important viruses. So far, this approach has successfully been applied to three of the 11 viral segments in our laboratory and others, and the efficiency of recovery of recombinant viruses has been improved. However, we are still waiting for the development of a helper virus‐free reverse genetics system for generating an infectious rotavirus entirely from cDNAs, as has been achieved for other members of the Reoviridae family.     

Analysis on the phenotype of E-FABP-gene knockout mice

The fatty acids are shown to be critical in the maintenance of the water permeability barrier that is ascribed to the lipids in the intracellular milieu of the cornified cell layer in the epidermis. In view of this importance in the skin, we examined the phenotype of epidermal fatty acid binding protein (E-FABP)-deficient mice. In spite of total lack of E-FABP expression in the various tissues of E-FABP deficient mice, these animals appeared normal in gross and histological examination. In Northern blot analysis for other FABPs, the gene expression of heart (H-)-type FABP is specifically elevated in the liver of neonatal heterozygous and homozygous mice, suggesting the functional compensation of H-FABP for E-FABP deficiency during their development. In functional analyses of the skin, the basal transepidermal water loss (TEWL) of the adult homozygous mice showed lower levels compared with the wild-type mice, and the impairment of recovery in TEWL was observed in the homozygous mice when the lipid barrier of the skin was disrupted by acetone. These results demonstrate that E-FABP is responsible for the water permeability barrier of the skin, although the molecular mechanism remains to be further elucidated.